Agrobacterium tumefaciens has been widely used for introduction of genes into plants for basic research as well as for generation of commercially used transgenic crops. For plant transformation, the gene of interest is placed between the left and right border repeats of Agrobacterium T-(transferred) DNA. Generally, the T-DNA region harboring the transgene is stably integrated into the plant genome by using an appropriate plant transformation protocol. T-DNA originates from the Agrobacterium Ti-(tumor inducing) plasmid, but because Ti-plasmids are large and difficult to manipulate, smaller T-DNA binary vectors are currently predominately used for generation of transgenic plants.
Despite the fact that Agrobacterium has been commonly used for plant transformation for more than two decades, some problems using this bacterium still remain: Agrobacterium-mediated transformation frequently results in unwanted high copy number T-DNA integration events. Agrobacterium-mediated transformation generally results in lower transgene copy numbers than do other transformation methods such as particle bombardment or polyethylene glycol-mediated transformation. However, multiple integration events, often coupled with inverted repeat T-DNA integration patterns, may affect the stability of transgene expression by silencing mechanisms. An additional problem with Agrobacterium-mediated transformation is the propensity for DNA sequences “outside” the T-DNA region to be carried along with or transferred independently of T-DNA and integrated into the plant genome. Integration of such vector “backbone” sequences can occur with high frequency. In one study, backbone sequences were detected in 75% of tested transgenic tobacco plants, and often the entire vector “backbone” is introduced into the plant genome. Although there is no evidence in the literature that the presence of vector backbone sequences affects transgene expression, T-DNA vector backbones usually harbor bacterial antibiotic resistance genes that can create governmental regulatory problems.
A current goal in agricultural biotechnology is to make plants with low (preferably single) transgene copy number, and no Agrobacterium T-DNA vector backbone sequence. A major goal of scientists generating transgenic plants is to have a single copy integrated transgene (less likely to have problems with silencing), and no T-DNA vector backbone integration (fewer regulatory problems). Therefore, methods and compositions to generate backbone-free, low transgene copy number plants are desired.